Introduction and Objective •The concept of Platform Technology (capturing, purification and polishing) has been successfully applied to the Mab purification (Figure 1). •The objective of this study was to assess whether a similar concept can be applied to the purification of E. Coli expressed soluble recombinant proteins in order to facilitate vaccine antigen purification process development. •A case study with a specific challenge will also be discussed (Part II) Davinder Chawla and Yan-Ping Yang Bio Process Research & Development, sanofi pasteur, Toronto Methods •Chromatographic methods such as Anion or cation exchange, Hydrophobic interaction chromatography, mixed mode chromatography methods were used. •AKTA Explorer was used for small-scale method development and processes were scaled-up using AKTA PILOT. •In-Process samples were tested using inline monitoring by UV, conductivity etc. •Fractions were analyzed by SDS-PAGE or BCA protein assay. Conclusions •Several protein purification processes were developed using different protein antigens expressed in E. coli expression systems. •A trend was observed in most of these purification schemes i.e. strong anion exchange such as Q column worked well as primary capture step and had enough capacity to capture the target protein where the expression in upstream was 1-3 g/L (Figure 1) •Binding and elution conditions of each protein including the buffers to purify these protein varied and were dependent on the individual characteristics of these proteins (data not shown). •The purity of in-process fractions after 1st unit operation was typically >50%. (Figure 2) Crude harvest Capturing or gross purification Intermediate purification Concentration / buffer exchange Polishing Target: 70-80% purity Target: >90% purity Target: 50% purity Figure 1: Typical Purification Process
Muscularis externa of mouse esophagus is composed of two skeletal muscle layers in the adult. But less attention is paid to the histogenesis of the muscularis externa of the esophagus, and controversies still exist about the developmental process and the spatio-temporal expression characteristics of muscle-specific proteins during the development of esophageal muscularis externa. To further probe into the developmental pattern of muscularis externa of the mouse esophagus and the expression characteristics of different muscle-specific proteins, immunohistochemical and terminal deoxyribonucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP)-digoxigenin nick-end labeling apoptotic staining methods are used to investigate the expression patterns of different muscle-specific proteins and to elucidate the relationship of these protein expressions with the development of muscularis externa of the mouse esophagus. Thus, an understanding of the developing esophageal muscularis externa may be important for developing therapeutic strategies for the treatment of human esophagus diseases. Serial sections of mouse embryos from embryonic day (ED) 12 to ED18, and full-length esophagi from postnatal first to 5th day were stained with monoclonal antibodies against α-smooth muscle actin (α-SMA), α-sarcomerical actin (α-SCA), desmin, and monoclonal anti-skeletal myosin (MHC), while apoptosis was determined using the terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick-end labeling assay. The expression of α-SMA was started at ED12. During the development of ED14-ED15, α-SMA positive cells were seen extending from the walls of left three, four, and six arch arteries toward the dorsal wall of esophagus. Stronger expression of α-SCA and desmin could be detected at ED14 and ED15, expression intensity in caudal segment and inner layer was stained stronger than that of cranial segment and outer layer, but after ED16, strong expression of α-SCA and desmin was found in the outer layer of muscularis externa. Expression of MHC was first detected in the outer layer of cranial segment of muscularis externa at ED17. At ED18, MHC had extended to the level of thyroid gland, staining intensity in the outer layer and cranial segment was stronger than that of inner layer and caudal segment. One to five days after birth, the thickness of the esophageal muscle layer was obviously increased. Most of the muscle cells in the cranial segment of esophagus showed strong expression of α-SCA and clear cross striations at higher magnification. With progression toward the caudal segment, expression intensity of α-SCA became weaker, but the expression intensity of desmin was the same at different levels of esophagus. The muscle fibers were arranged densely with high expression of MHC in the cranial segment. During the development of esophageal muscularis externa, few apoptotic cells were observed. α-SMA, α-SCA, desmin, and MHC show different expression patterns. The differentiation of outer layer of esophageal muscularis externa is quicker than that of inner layer, and the caudal segment is quicker than that of the cranial segment. Besides, apoptosis may not participate in the development of esophageal muscularis externa. The smooth muscle cells from arch arteries may participate in the development of esophageal muscularis externa.
The second heart field ( SHF ), foregut endoderm and sonic hedgehog ( SHH ) signaling pathway are all reported to associate with normal morphogenesis and septation of outflow tract ( OFT ). However, the morphological relationships of the development of foregut endoderm and expression of SHH signaling pathway members with the development of surrounding SHF and OFT are seldom described. In this study, serial sections of mouse embryos from ED 9 to ED 13 (midgestation) were stained with a series of marker antibodies for specifically highlighting SHF (Isl‐1), endoderm (Foxa2), basement membrane (Laminin), myocardium ( MHC ) and smooth muscle ( α ‐ SMA ) respectively, or SHH receptors antibodies including patched1 (Ptc1), patched2 (Ptc2) and smoothened, to observe the spatiotemporal relationship between them and their contributions to OFT morphogenesis. Our results demonstrated that the development of an Isl‐1 positive field in the splanchnic mesoderm ventral to foregut, a subset of SHF , is closely coupled with pulmonary endoderm or tracheal groove, the Isl‐1 positive cells surrounding pulmonary endoderm are distributed in a special cone‐shaped pattern and take part in the formation of the lateral walls of the intrapericardial aorta and pulmonary trunk and the transient aortic‐pulmonary septum, and Ptc1 and Ptc2 are exclusively expressed in pulmonary endoderm during this Isl‐l positive field development, suggesting special roles played in inducing the Isl‐l positive field formation by pulmonary endoderm. It is indicated that pulmonary endoderm plays a role in the development and specification of SHF in midgestation, and that pulmonary endoderm‐associated Isl‐l positive field is involved in patterning the morphogenesis and septation of the intrapericardial arterial trunks.
To the Editor: Elephantiasis results from chronic lymphedema and is characterized by gross enlargement of the arms, legs, or genitalia. It occurs due to various obstructive diseases of the lymphatic system. The most common form of lymphedema is secondary lymphedema involving resection or ablation of the regional lymph nodes by surgery, radiation, tumor invasion, direct trauma, or an infection.[12] We present an unusual case of bilateral lower limb and abdominal elephantiasis due to extensive lymph node destruction by erysipelas. A 31-year-old man presented with progressively increasing bilateral lower limb and abdominal swelling, with changes of skin over 1 year. He also described a history of repeated outbreaks of erysipelas in the right leg 5 years prior to this presentation, for which he had multiple hospital admissions and received treatment with intravenous antibiotics, whose names he could not recollect. Physical examination revealed that he was obese, weighing about 244 kg, with a body mass index of 75.3 kg/m2. The patient had giant-sized bilateral lower limbs and abdominal swelling, with a chronic disseminated dermatosis of the skin, characterized by edema, hyperpigmentation, hyperkeratosis, and elephantiasis nostras verrucosa (ENV) [Figure 1a and 1b]. His blood lipid parameters and serum cortisol were normal, without microfilaria.Figure 1: Chronic lymphedema in lower extremities and abdomen (a), and hyperpigmentation, hyperkeratosis, and a verrucous aspect involving the abdomen (b). Vascular ultrasound examination of the lower limb shows the sizes (1.06 cm × 1.92 cm) (c) and no significant blood flow (d) in the swollen lymph node in the left groin. Histopathological examination of the specimen of swollen lymph node in the left groin. The features were suggestive of nonspecific inflammation of lymph node cortical atrophy, lymphatic sinus dilation, and interstitial vascular proliferation with dilation (e and f) (H and E staining, original magnification ×200).The patient underwent vascular ultrasound examination of lower limbs, which showed swollen lymph nodes in bilateral inguinal region [Figure 1c and 1d]. A histopathological examination of the specimen showed lymph node changes in the left inguinal region. The features were suggestive of nonspecific inflammation of lymph node, cortical atrophy, lymphatic sinus dilation, and interstitial vascular proliferation with dilation [Figure 1e and 1f]. However, there was no clear evidence of erysipelas, malignancy, filariasis, or donovanosis in the specimens. The term “elephantiasis” describes an elephant-like appearance or overt enlargement of the legs, arms, or vulva.[3] Lymphedema manifests as soft-pitting edema in the affected tissues that results in a local inflammatory response, which finally leads to nonpitting edema. The affected tissues sustain further injury as a result of the local inflammatory response and recurrent infections. The common mechanism is an underlying lymphatic obstruction leading to impaired lymphatic drainage with abnormal accumulation of interstitial fluid and subsequent development of lymphedema. This eventually results in excessive subcutaneous fibrosis and scarring, with associated severe skin changes characteristic of lymphostatic elephantiasis.[2] ENV is a rare clinical condition associated with chronic nonfilarial lymphedema caused by bacterial or noninfectious lymphatic obstruction. Mossy papules, plaques, and cobblestone-like nodules are clinical features of ENV.[4] The patient's history and characteristic skin changes are typically sufficient to diagnose ENV. Our patient with bilateral lower limb elephantiasis and abdominal ENV is a very rare case. The absence of erysipelas histology from the lymph node ruled out direct infiltration. Moreover, the patient had a past history of repeated erysipelas outbreaks. The etiology in our case was extensive destruction of the inguinal lymph nodes and their channels due to past erysipelas outbreaks, leading to a blockage of lymphatic drainage, resulting in lower limb elephantiasis and abdominal ENV. Many factors, including surgery, radiation, or an infection, may lead to chronic lymphatic obstruction and stasis. Filariasis, caused by infestation of the lymph nodes by the parasite Wuchereria bancrofti, is the most common and global cause of secondary lymphedema.[2] The precise role of some pathogens in lymphatic obstruction is uncertain. Treatment options of elephantiasis include use of elastic bandages, pneumatic stockings, mechanical massage, oral retinoids, and surgery. Reconstructive surgery is often considered to be the only treatment for serious penoscrotal elephantiasis.[5] The goal of the therapy is to re-establish function and reduce physical disability. Declaration of patient consent The authors certify that they have obtained all appropriate patient consent forms. In the form, the patient has given his consent for his images and other clinical information to be reported in the journal. The patient understands that his name and initials will not be published and due efforts will be made to conceal his identity, but anonymity cannot be guaranteed. Financial support and sponsorship This work was supported by a grant from the Foundation of Shanghai Jiao Tong University Affiliated Sixth People's Hospital (No. ynlc201709). Conflicts of interest There are no conflicts of interest.
A 43-year-old Chinese man presented with generalized hypohidrosis, which he had had since birth, without obvious abnormalities of other skin appendages except a sparse beard and axillary hairs. The sweat test revealed localized sweating on the face, axillae and palms. Histopathologic examination showed that the sweat glands were absent in the forearm and thigh, but some eccrine and apocrine sweat glands were present in the right axilla. S-100 was expressed in the nerve terminals surrounding the acini and ducts of the eccrine sweat glands, while PGP9.5 was positive in the acini of apocrine glands and the nerve terminals surrounding the eccrine glands in the axilla. To our knowledge, this is the first case of congenital idiopathic hypohidrosis in China.
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